Specific association of complementary nucleic acid takes place in a reaction commonly referred to as hybridization. This technique has made possible the sensitive and specific detection, and the isolation, of nucleic acid sequences. In this technique, one prepares a nucleic acid probe which is complementary to a target nucleic acid, and then by selecting the appropriate conditions causes these two entities to bind to each other to form a hybrid. By judiciously choosing the reaction conditions, e.g. temperature, ionic strength and the like, one prevents non-complementary nucleic acids from hybridizing. Hybrids can be formed on a solid support, in tissue sections or in solution. In the last case they are subsequently separated from a mixture of nucleic acids by one of a variety of methods and immobilized on a solid support. The formation of hybrids can be followed if a "reporter" probe contains a detectable element, such as a radioactive atom, an enzyme with detectable activity, a hapten to which an enzyme-conjugated antibody can bind, or generally any entity to which an enzyme-conjugated ligand can bind.
Since its discovery, nucleic acid hybridization has been used extensively as a research tool. More recently, it has been put to use as a medical diagnostic technique, as a method for testing food for the presence of pathogens, in agriculture, etc. In these fields, hybridization is in its infancy: there is an ongoing need to develop improved techniques, to make the assay methods more sensitive, more specific, faster and easier to use. It is one aspect of the present invention to provide an improved reagent for use in hybridization assays.
Most hybridization assays are done overnight with targets immobilized on filters (Nygaard, A. P. and Hall, B. D. (1963) Biochem. Biophys. Res. Commun. 12, 98-104; Gillespie, D. and Spiegelman, S. (1965) J. Molec. Biol. 12, 829-842; Southern, E. M. (1975) J. Molec. Biol. 98, 503-517). Though these techniques have been extremely useful for research purposes and have made possible a variety of significant discoveries, a suitable clinical assay must be much more rapid (with results available within an hour or two of specimen collection), as well as being simpler (filter immobilization is not trivial, especially with clinical samples) and more sensitive.
The nucleic acids in a hybridization reaction generally have to be highly purified prior to immobilization, especially when non-radioactive detection is used (Ruth, J. L. and Bryan, R. N. (1984) Fed. Proc. 43, 2048; Kuritza, A. P., Getty, C. E., Shaughnessy, P., Hesse, R. and Salyers, A. A. (1986) J. Clin. Microbiol. 23, 343-349; Zwadyk, P., Cooksey, R. C. and Thornsberry, C. (1986) Curr. Microbiol. 14, 95-100). While this is less of a major problem in a research setting, this is unacceptable in a clinical situation because nucleic acid purification can be a multi-step, time-consuming process, where an assay must be rapid and simple.
It is another aspect of the present invention to provide a clinical assay incorporating a rapid and simplified sample preparation step along with reagents suitable therefor. Gillespie (International Application No. WO 87/06621, dated 5 Nov. 1987) has described a method of carrying out nucleic acid hybridizations which does not require prior purification and/or immobilization of solubilized target nucleic acids. In this method, guanidinium thiocyanate serves both to solubilize a target nucleic acid and to permit hybridization. While this technique overcame some of the problems associated with the development of a rapid, simple and versatile nucleic acid hybridization assay appropriate for use in a clinical setting, the technique shared many of the same limitations inherent in other hybridization procedures. For example, there remained an absolute limit on the extent of nucleic acid labeling by hybridization: at most, one complementary reporter probe can bind to each accessible binding site on the target molecule. Furthermore, as with other methods, hybridization rates and optimal hybridization temperatures depend on the target sequence's (G+C) content, a parameter which can vary considerably (discussed in detail below).
It is still another aspect of the present invention to provide reagents which do not depend upon the G+C content of target sequences as do conventional reagents.
When highly impure samples have been immobilized on filters, detection has been limited for the most part to radioactive methods (Bresser, J., Doering, J. and Gillespie, D. (1983) DNA 2, 243-254; Fitts, R. (1985) Food Technology 39, 95-102). This presents a significant problem for a clinical laboratory, since many are not licensed to work with radioactive materials, some personnel have little or no experience working with radioactive materials, work with radioactive materials involves some health risk, and radioactivity decays with time (in the case of certain isotopes, quite rapidly) and can cause radiolysis of probes, thus limiting the usable lifetimes of these materials and reducing the feasibility of a clinical assay employing the materials. Non-radioactive detection methods have been developed (for example, see Leafy, J. J., Brigati, D. J. and Ward, D. C. (1983) Proc. Natl. Acad. Sci. USA 80, 4045-4049), but their sensitivity has not matched that of the radioactive methods except where the target was highly purified prior to hybridization and detection.
`Sandwich hybridization` assays were developed (Dunn, A. R. and Hassell, J. A. (1977) Cell 12, 23-36; Ranki, M., Palva, A., Virtanen, M., Laaksonen, M. and Soderlund, H. (1983) Gene 21, 77-85; Virtanen, M., Palva, A., Laaksonen, M., Halonen, P., Soderlund, H. and Ranki, M. (1983) Lancet 1, 383-393) in an attempt to overcome some of the limitations of conventional hybridization techniques. The target nucleic acid is `sandwiched` between a capture probe immobilized on a solid support and a labeled probe which is complexed with the target in solution. Though the `sandwich` technique represented an improvement in some aspects of nucleic acid hybridizations, the procedure still retained certain weaknesses, notably its slowness, its clinically insufficient sensitivity and its seemingly inherent background problems thus lowering sensitivity and specificity.
Various techniques have been developed to accelerate the hybridization process. For example the two-phase phenol/aqueous emulsion procedure (Kohne, D. E., Levinson, S. A., and Byers, M. J. (1977), Biochemistry 16, 5329) has been reported to result in reaction rates over 100 times faster than reference rates. Deficiencies of this method, however, are that it fails to boost reaction rates to the same extent when RNA is involved, that it requires that the reaction vessel be agitated during the hybridization process, and that it involves a chemical (phenol) associated with certain health risks.
It is still another aspect of the present invention to provide reagents which are effective in improving RNA hybridization rates.
It is still yet another aspect of the present invention to provide assays which overcome some of the deficiencies of conventional assays.
It is yet still another aspect of the present invention to provide reagents which do not pose the health risks associated with phenol.
Another technique reported to accelerate hybridization reactions is to add to a reaction mixture volume exclusion reagents, such as polyethylene glycol (Renz and Kurz (1984), Nucl. Acids Res., 12, 3435-3444), dextran or dextran sulfate (Wetmur, J. G. (1975), Biopolymers 14, 2517-2524; Wahl, G. M., Stern, M., and Stark, G. R. (1979), Proc. Natl. Acad. Sci. USA 76, 3683-3687; Wahl, G. M. and Stark, G. R., U.S. Pat. No. 4,302,204, Nov. 24, 1981), polyacrylate or polymethacrylate (Boguslawski, S. J. and Anderson, L. H. D., U.S. Pat. No. 4,689,294, Aug. 25, 1987). Though these methods accelerate hybridization when a target is immobilized the acceleration is less dramatic in solution hybridizations. Furthermore, while these techniques increase hybridization rates, they do not raise final hybridization levels.
It is still further an aspect of the present invention to provide reagents which increase hybridization rates in solution hybridization.
It is another still further aspect of the present invention to provide reagents which raise final hybridization levels.
Kohne and Kacian (European Patent Application number 86304429.3) have also reported a method of accelerating nucleic acid hybridization using different agents that also precipitate nucleic acids. In their application, the claims are limited to increased rates of nucleic acid reassociation, with no mention whatsoever of increased levels (i.e., final signals). There remains the labeling limit of, at most, one complementary reporter probe per binding site on the target molecule. Additionally, these investigators do not disclose any nucleic acid precipitation agents which possess capabilities such as inactivating nucleases, allowing proteases to function effectively, or strengthening binding, all which are further aspects to be provided by the reagents of the present invention.
Numerous modifications have been made to the original sandwich hybridization format, including replacing the filter with other solid supports (Rashtchian, A., Eldredge, J., Ottaviani, M., Abbott, M., Mock, G., Lovern, D., Klinger, J. and Parsons, G. (1987) Clinical Chemistry 33, 1526-1530) Langdale, J. A. and Malcolm, A. D. B. (1985) Gene 36, 210-220) and the use of affinity methods to improve the speed of the capture process (Rashtchian et al. (1987); Langdale and Malcolm (1985); Langer, P. R., Waldrop, A. A. and Ward, D. C. (1981) Proc. Natl. Acad. Sci. USA 78, 6633-6637; Manning, J., Pellegrini, M. and Davidson, N. (1977) Biochemistry 16, 1364-1370; Delius, H., van Heerikhuzen, H., Clarke, J. and Koller, B. (1985) Nucl. Acids Res. 13, 5457-5469; Dale, R. M. K. and Ward, D. C. (1975) Biochemistry 14, 2458-2469; Banfalvi, G., Bhattacharya, S. and Sarkar, N. (1985) Anal. Biochem. 146, 64-70; Arsenyan, S. G., Avdonia, T. A., Laving, A. Saarma, M. and Kisselev, L. L. (1980) Gene 11, 97-108). These formats all employ a single capture step. Insufficient specificity and sensitivity, however, remain problems for the clinical application of these techniques.
Another yet further aspect of the present invention is to provide assays employing new reagents which provide the necessary specificity and sensitivity for the clinical application of hybridization assays.
Another problem with the general application of conventional hybridization techniques results from the fact that different target nucleic acid sequences (and the corresponding complementary probes) vary considerably in (G+C) content. Consequently, since the hybrid melting temperature and the optimal hybridization temperature in commonly used assay reagents vary as a function of the (G+C) content, they will be different for target:probe combinations of different composition (assuming the sequence lengths are the same). This means that the optimal assay temperature in the standard assay reagents may be different for each assay. This is highly undesirable since it makes it very difficult to standardize the assay format to allow for the testing of many samples for different organisms in a common instrument at a single temperature. Although probe length can be varied somewhat to correct for differences in melting temperatures (among probes with widely differing (G+C) content), inclusivity and exclusivity requirements set practical limits on this approach.
It is another aspect of the present invention to provide regents and methods which employ a general assay protocol, e.g. temperature, for a wide range of tests which is unavailable with hybridization media in common use.
Melchior and von Hippel (Proc. Natl. Acad. Sci. USA, 70 (1973), 298-302) have observed that in solutions containing tetraalkylammonium cations the A:T base-pair is strengthened relative to the G:C basepair. Hamaguchi and Geiduschek (Jour. Amer. Chem. Soc. 84 (1962), 1329-1338) have reported that chaotropic anions denature nucleic acids and disrupt the A:T base pair more strongly than the G:C base-pair. These references, however, fail to disclose how the results may be utilized to provide reagents meeting the various aspects of the present invention and in particular fail to teach whether achieving GC/AT equivalence is desirable and if so, how it is to be achieved.
It is yet another aspect to overcome the limit of one reporter probe binding to each target site inherent in all heretofore available hybridization procedures.
It is still another aspect of the present invention to dramatically increase the level of signals that one can obtain in traditional hybridization assays.
All documents cited herein are fully incorporated herein by reference.